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EP2956538B1 - Constructions hépatiques biologiquement modifiées et méthodes associées - Google Patents

Constructions hépatiques biologiquement modifiées et méthodes associées Download PDF

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EP2956538B1
EP2956538B1 EP14706251.7A EP14706251A EP2956538B1 EP 2956538 B1 EP2956538 B1 EP 2956538B1 EP 14706251 A EP14706251 A EP 14706251A EP 2956538 B1 EP2956538 B1 EP 2956538B1
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cells
liver
hsc
cell
derived
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EP2956538A1 (fr
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Maria Graca ALMEIDA-PORADA
Pedro Miguel A.M. BAPTISTA
Shay Soker
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Wake Forest University Health Sciences
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Definitions

  • the present invention concerns to bioengineered liver constructs, methods of making such constructs, and method of using such constructs.
  • WO2012/005760 describes a 3D organ- or tissue-specific ECM scaffold, e.g., for matched organ- or tissue-specific differentiation or maturation, or maintenance, of cell types including stem or progenitor cells, or differentiated or primary cells.
  • Tissue engineering strategies have been developed for the generation of functional liver tissue using decellularized liver scaffolds repopulated with liver cells [9-12, 70-72, 78].
  • the decellularized liver bioscaffold offers several advantages over widely used synthetic polymer 3D scaffolds.
  • the preservation of intact native vascular channels can be used to deliver cells into the parenchyma, culture media with nutrients and oxygen to the newly generated tissue, and subsequently anastomosed to the host vascular system.
  • the retention of important ECM molecules in their native configuration can allow for site specific engraftment of different cell types along with providing signals that direct polarization, migration, proliferation, survival and differentiation [9-11].
  • Human fetal liver progenitor cells (hFLPCs) isolated from fetal livers can be used as a recellularized cell source due to their capability of differentiating into hepatocytes and cholangiocytes [9, 10].
  • HSC multipotent hematopoietic stem cells
  • HSC tissue-specific stem cells that exhibit remarkable proliferative and self-renewal capacity and are responsible for the life-long maintenance of the hematopoietic system.
  • HSCs are rare cells that reside in adult bone marrow where hematopoiesis is continuously taking place. They can also be found in cord blood, fetal liver, adult spleen, and peripheral blood.
  • HSC transplantation provides the only curative treatment for a wide variety of malignant and nonmalignant hematologic disorders (e.g., bone marrow failure states such as myelodysplastic syndrome, metabolic storage diseases, and hemoglobinopathies).
  • HSCT are preferred over transplantations using more differentiated cells (e.g., hematopoietic progenitor cells (HPC)), which have an increased risk of failure long-term.
  • HPC hematopoietic progenitor cells
  • HPC hematopoietic progenitor cells
  • HSCTs are either autologous (using the subject's own cells) or allogeneic (using donor stem cells, typically from matched donor).
  • HSC bone marrow
  • peripheral blood and umbilical cord blood
  • CB umbilical cord blood
  • PBSC peripheral blood stem cells
  • G-CSF granulocyte colony stimulating factor
  • CD34 CD34-positive progenitors and total cells than bone marrow
  • peripheral blood stem cells are the most common source of HSCs for allogeneic HSCT.
  • HLA human leukocyte antigen
  • HSC hematopoietic stem cell
  • HSCs must be able to expand on a larger scale without sacrificing their self-renewal ability; and (2) expanded HSC must be safe and transplantable, and requires the method to be free of feeder cells, serum proteins, or microbial agents.
  • HSC expansion is challenging due to the fact that culturing HSC results in differentiation of the cells into progenitor cells in the hematopoiesis lineages. Although HSC will rapidly expand after in vivo transplantation, experience from in vitro studies indicates that control of HSC self-renewal and differentiation in culture remains difficult.
  • CB-HSPC can be expanded and the differentiation of these cells can be driven towards both the myeloid and lymphoid lineages in a serum-free culture system using a feeder layer of adult human bone marrow-derived stromal cells [3-6].
  • Initial progenitor content and cytokine concentrations for ex vivo expansion in 2-D cultures have also be assessed [6-8], and demonstrated that the expanded cells had the ability to engraft pre-immune fetal sheep [4].
  • One aspect of the invention is a method of generating a liver organoid comprising the steps of:
  • liver organoid comprising a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels; and liver cells having predominately fetal characteristics.
  • the liver cells may comprise at least one of liver progenitor cells, hepatoblasts, vascular cells, cholangiocytes, or stromal cells.
  • the vascular cells may comprise at least one of liver endothelial cells, liver sinusodial cells, vascular smooth muscle cells, or pericytes.
  • the hepatoblasts may comprise at least one of fetal liver hepatoblasts, hepatoblasts derived from adult-derived liver stem cells, hepatoblasts derived from induced pluripotent stem cells, or hepatoblasts derived from embryonic stem cells.
  • the liver cells may comprise an enriched fetal liver progenitor cell population comprising at least about 55-75 % hepatic cells and not more than about 15-25 % stromal cells and not more than about 5-15 % endothelial cells.
  • the liver cells may comprise an enriched fetal liver progenitor cell population comprising about 55-75 % hepatic cells, about 15-25 % stromal cells, about 5-15 % endothelial cells.
  • the liver cells may express at least one of ⁇ -fetoprotein or CYP3A7.
  • the hepatic cells may comprise fetal liver progenitor cells.
  • liver organoid comprising a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels; and liver cells having adult characteristics.
  • the liver cells may comprise at least one of hepatoblasts, hepatocytes, vascular cells, cholangiocytes, or stromal cells.
  • the vascular cells may comprise at least one of liver endothelial cells, liver sinusodial cells, vascular smooth muscle cells, or pericytes.
  • the hepatocytes may be derived from at least one of fetal liver hepatoblasts, hepatoblasts derived from adult-derived liver stem cells, hepatoblasts derived from induced pluripotent stem cells, or hepatoblasts derived from embryonic stem cells.
  • the liver organoid may comprise hepatocytic cell clusters and biliary ducts.
  • the liver cells may express albumin.
  • the liver cells may express at least one cytochrome P450 isoform.
  • the liver cells may express at least one of CK7, CK19, transferrin, CYP3A4, HNF4 ⁇ , AST, ALT, TAT, CYP2E1 or A1AT.
  • the liver cells may synthesize urea.
  • liver organoid comprising the steps of providing a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels, seeding the bioscaffold with liver cells; and culturing the liver cells with culture medium containing at least one growth factor to generate a liver organoid comprising differentiated liver cells over time.
  • the liver cells may comprise at least one of hepatoblasts, hepatocytes, vascular cells, cholangiocytes, or stromal cells.
  • the vascular cells may comprise at least one of liver endothelial cells, liver sinusodial cells, vascular smooth muscle cells, or pericytes.
  • the hepatoblasts may comprise at least one of fetal liver hepatoblasts, hepatoblasts derived from adult-derived liver stem cells, hepatoblasts derived from induced pluripotent stem cells, or hepatoblasts derived from embryonic stem cells.
  • the liver cells of step (b) may comprise an enriched fetal liver progenitor cell population comprising at least about 55-75 % hepatic cells and not more than about 15-25 % stromal cells and not more than about 5-15 % endothelial cells.
  • the liver cells of step (b) may comprise an enriched fetal liver progenitor cell population comprising about 55-75 % hepatic cells, about 15-25 % stromal cells, about 5-15 % endothelial cells.
  • the hepatic cells may comprise fetal liver progenitor cells.
  • the liver organoid may comprise hepatocytic cell clusters and biliary ducts.
  • the differentiated liver cells of step (c) may express albumin.
  • the differentiated liver cells of step (c) may express at least one cytochrome P450 isoform.
  • the culture medium of step (c) may comprise oncostatin M.
  • the culture medium of step (c) may comprise at least one of dexamethasone, cyclic adenosine monophosphate (cAMP), Prolactin, Glucagon, niacinamide, ⁇ -lipoic acid, triiodothyronine, free fatty acids, epidermal growth factor (EGF), high-density lipoprotein (HDL), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), or human growth hormone (HGH).
  • cAMP cyclic adenosine monophosphate
  • Prolactin Prolactin
  • Glucagon Prolactin
  • niacinamide ⁇ -lipoic acid
  • triiodothyronine triiodothyronine
  • free fatty acids EGF
  • EGF epidermal growth factor
  • HDL high-density lipoprotein
  • HGF hepatocyte growth factor
  • FGF fibroblast growth factor
  • HGH human growth
  • liver organoid comprising a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels; and liver cells comprising at least one micro-environment niche that supports hematopoietic stem cell (HSC) expansion or differentiation.
  • the at least one micro-environment niche may support expansion or differentiation of at least one of HSC obtained from cord blood, HSC obtained from bone marrow, HSC derived from induced pluripotent stem cells, HSC derived from embryonic stem cells, or HSC derived from direct reprogramming of adult somatic cells.
  • the liver cells of the liver organoid comprise at least two micro-environment niches, wherein in at least one micro-environment niche supports HSC expansion and at least one micro-environment niche supports HSC differentiation.
  • the liver cells of the liver organoid may be fetal liver cells.
  • the liver cells may be at least one of liver progenitor cells, hepatoblasts, vascular cells, cholangiocytes, or stromal cells.
  • the vascular cells may be at least one of liver endothelial cells, liver sinusoidal cells, smooth muscle cells, or pericytes.
  • the hepatoblasts may be at least one of fetal liver hepatoblasts, hepatoblasts derived from adult-derived liver stem cells, hepatoblasts derived from induced pluripotent stem cells, or hepatoblasts derived from embryonic stem cells.
  • the at least one micro-environment niche supports expansion or differentiation of at least one of HSC obtained from cord blood, HSC obtained from bone marrow, HSC derived from induced pluripotent stem cells, HSC derived from embryonic stem cells, or HSC derived from direct reprogramming of adult somatic cells.
  • the method includes the steps of: providing a bioscaffold derived from a decellularized donor subject liver comprising an extracellular matrix (ECM) and native vascular channels; seeding the bioscaffold with liver cells; culturing the liver cells with the bioscaffold in the presence of culture media for sufficient time to produce at least one micro-environment niche in the bioscaffold that supports hematopoietic stem cell (HSC) expansion or differentiation.
  • ECM extracellular matrix
  • HSC hematopoietic stem cell
  • the liver cells are cultured with the bioscaffold in the presence of culture media for sufficient time to produce at least two micro-environment niches in the bioscaffold, wherein at least one micro-environment niche supports HSC expansion and at least one micro-environment niche supports HSC differentiation.
  • the liver cells of the liver organoid are as described in the previous paragraph.
  • the liver cells seeded on the organoids comprise an enriched fetal liver progenitor cell population containing at least about 55-75 % hepatic cells, not more than about 15-25 % stromal cells and not more than about 5-15 % endothelial cells.
  • these liver cells comprise an enriched fetal liver progenitor cell population containing about 55-75 % hepatic cells, about 15-25 % stromal cells, and about 5-15 % endothelial cells.
  • the hepatic cells may comprise fetal liver progenitor cells.
  • the liver cells seeded on the organoid may be cultured in culture media comprising at least one of epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), glycogen synthase kinase-3beta inhibitor (GSK3 ⁇ i), or thiazovivin.
  • EGF epidermal growth factor
  • HGF hepatocyte growth factor
  • IGF-1 insulin-like growth factor 1
  • GSK3 ⁇ i glycogen synthase kinase-3beta inhibitor
  • the at least one micro-environment niche may support expansion or differentiation of at least one of HSC obtained from cord blood, HSC obtained from bone marrow, HSC derived from induced pluripotent stem cells, HSC derived from embryonic stem cells, or HSC derived from direct reprogramming of adult somatic cells.
  • the liver cells seeded on the organoid may be cultured for about 5 days.
  • the culture media for culturing the HSC cells may contain at least one exogenous factor.
  • the media contains at least one of stem cell factor (SCF), interleukin-6 (IL-6), or Fms-like tyrosine kinase 3 (FLT3), oncostatin-M (OSM), TGF ⁇ 3, Jagged-1, fetal bovine serum (FBS), dexamethasone, cyclic adenosine monophosphate (cAMP), Prolactin, Glucagon, niacinamide, ⁇ -lipoic acid, triiodothyronine, epidermal growth factor (EGF), high-density lipoprotein (HDL), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), or human growth hormone (HGH).
  • SCF stem cell factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • OSM Fms-like tyrosine kinase 3
  • the media may comprise at least one of stem cell factor (SCF), fibroblast growth factor (FGF), interleukin-6 (IL-6), Fms-like tyrosine kinase 3 (FLT3), or leukemia inhibitory factor (LIF).
  • SCF stem cell factor
  • FGF fibroblast growth factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • LIF leukemia inhibitory factor
  • factors added to the culture media are human factors.
  • the liver organoid is seeded with at least one of HSC obtained from cord blood, HSC obtained from bone marrow, HSC derived from induced pluripotent stem cells, HSC derived from embryonic stem cells, or HSC derived from direct reprogramming of adult somatic cells.
  • the differentiated hematopoietic cells may comprise hematopoietic progenitor cells (HPC), myeloid progenitor cells (MPC), lymphoid progenitor cells (LPC), lymphocytes, granulocytes, macrophages, erythrocytes, or platelets.
  • HPC hematopoietic progenitor cells
  • MPC myeloid progenitor cells
  • LPC lymphoid progenitor cells
  • lymphocytes granulocytes
  • macrophages erythrocytes
  • platelets erythropoietin
  • the method includes the steps of: obtaining a liver organoid comprising a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels and liver cells comprising at least one micro-environment niche that supports hematopoietic stem cell (HSC) expansion or differentiation; seeding the liver organoid with HSC; culturing the HSC on the liver organoid with culture media; and collecting expanded HSC and/or differentiated hematopoietic cells from the culture media.
  • ECM extracellular matrix
  • HSC hematopoietic stem cell
  • the liver organoid comprises (i) a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels and (ii) liver cells comprising at least two micro-environment niches, wherein at least one micro-environment niche supports HSC expansion and at least one micro-environment niche supports HSC differentiation.
  • the method enables collection of differentiated hematopoietic cells, including hematopoietic progenitor cells (HPC), myeloid progenitor cells (MPC), lymphoid progenitor cells (LPC), lymphocytes, granulocytes, macrophages, erythrocytes, or platelets.
  • HPC hematopoietic progenitor cells
  • MPC myeloid progenitor cells
  • LPC lymphoid progenitor cells
  • the culture media does not include growth factors and/or cytokines.
  • the culture media may exclude growth factors when expanded HSC and/or immature hematopoietic stem cells are collected from the culture media.
  • the culture media that excludes growth factors may contain at least one of stem cell factor (SCF), fibroblast th factor (FGF), mterleukin-6 (IL-6), Fms-like tyrosine kinase 3 (FLT3), or leukemia inhibitory factor (LIF).
  • SCF stem cell factor
  • FGF fibroblast th factor
  • IL-6 mterleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • LIF leukemia inhibitory factor
  • the culture media may contain at least one growth factor when differentiated hematopoietic stem cells, such as, e.g., hematopoietic progenitor cells (HPC), myeloid progenitor cells (MPC), lymphoid progenitor cells (LPC), lymphocytes, granulocytes, macrophages, erythrocytes, or platelets, are collected in from the culture media.
  • HPC hematopoietic progenitor cells
  • MPC myeloid progenitor cells
  • LPC lymphoid progenitor cells
  • the culture media containing at least one growth factor may contain at least one of stem cell factor (SCF), interleukin-6 (IL-6), or Fms-like tyrosine kinase 3 (FLT3), oncostatin-M (OSM), TGF ⁇ 3, Jagged-1, fetal bovine serum (FBS), dexamethasone, cyclic adenosine monophosphate (cAMP), Prolactin, Glucagon, niacinamide, ⁇ -lipoic acid, triiodothyronine, epidermal growth factor (EGF), high-density lipoprotein (HDL), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), or human growth hormone (HGH).
  • SCF stem cell factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • OSM oncostatin-M
  • the culture media may contain at least one of stem cell factor (SCF), fibroblast growth factor (FGF), interleukin-6 (IL-6), Fms-like tyrosine kinase 3 (FLT3), or leukemia inhibitory factor (LIF).
  • SCF stem cell factor
  • FGF fibroblast growth factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • LIF leukemia inhibitory factor
  • an enriched population of differentiated erythrocytes is collected.
  • the culture media will at least include erythropoietin (EPO).
  • EPO erythropoietin
  • the culture media is serum-free culture media.
  • the liver organoid may be seeded with at least one of HSC obtained from cord blood, HSC obtained from bone marrow, HSC derived from induced pluripotent stem cells, HSC derived from embryonic stem cells, or HSC derived from direct reprogramming of adult somatic cells.
  • a cell population comprising hematopoietic cells produced by a process comprising (a) obtaining a liver organoid comprising (i) a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels and (ii) liver cells comprising at least one micro-environment niche that supports hematopoietic stem cell (HSC) expansion or differentiation, (b) seeding the liver organoid with HSC, (c) culturing the HSC on the liver organoid with culture media; and (d) collecting hematopoietic cells from the culture media.
  • a liver organoid comprising (i) a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels and (ii) liver cells comprising at least one micro-environment niche that supports hematopoietic stem cell (HSC) expansion or differentiation
  • HSC hema
  • the hematopoietic cells may comprise expanded HSC and/or differentiated hematopoietic cells.
  • the organoid may comprise (i) a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular network and (ii) liver cells comprising at least two micro-environment niches, wherein at least one micro-environment niche supports HSC expansion and at least one micro-environment niche supports HSC differentiation.
  • the liver cells of step (a) may comprise fetal liver cells.
  • the liver cells of step (a) may comprise at least one of liver progenitor cells, hepatoblasts, vascular cells, cholangiocytes, or stromal cells.
  • the vascular cells may comprise at least one of liver endothelial cells, liver sinusodial cells, vascular smooth muscle cells, or pericytes.
  • the hepatoblasts may comprise at least one of fetal liver hepatoblasts, hepatoblasts derived from adult-derived liver stem cells, hepatoblasts derived from induced pluripotent stem cells, or hepatoblasts derived from embryonic stem cells.
  • the liver cells of step (a) may comprise an enriched fetal liver progenitor cell population comprising at least about 55-75 % hepatic cells and not more than about 15-25 % stromal cells and not more than about 5-15 % endothelial cells.
  • the liver cells of step (a) may comprise an enriched fetal liver progenitor cell population comprising about 55-75 % hepatic cells, about 15-25 % stromal cells, about 5-15 % endothelial cells.
  • the hepatic cells may comprise fetal liver progenitor cells.
  • the liver cells seeded on the bioscaffold in step (b) may be cultured in culture media comprising at least one of epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), glycogen synthase kinase-3beta inhibitor (GSK3 ⁇ i), or thiazovivin.
  • EGF epidermal growth factor
  • HGF hepatocyte growth factor
  • IGF-1 insulin-like growth factor 1
  • GSK3 ⁇ i glycogen synthase kinase-3beta inhibitor
  • the liver cells seeded on the bioscaffold in step (b) may be cultured for about 5 days.
  • the culture media in step (c) may exclude exogenous growth factors, and wherein expanded HSC and/or immature hematopoietic stem cells are collected in step (d).
  • the culture media in step (c) may comprise at least one of stem cell factor (SCF), fibroblast growth factor (FGF), interleukin-6 (IL-6), Fms-like tyrosine kinase 3 (FLT3), or leukemia inhibitory factor (LIF).
  • SCF stem cell factor
  • FGF fibroblast growth factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • LIF leukemia inhibitory factor
  • the culture media in step (c) may comprise at least one exogenous growth factor, and wherein differentiated hematopoietic stem cells are collected in step (d).
  • the culture media in step (c) may comprise at least one of stem cell factor (SCF), interleukin-6 (IL-6), Fms-like tyrosine kinase 3 (FLT3), oncostatin-M (OSM), TGF ⁇ 3, Jagged-1, fetal bovine serum (FBS), dexamethasone, cyclic adenosine monophosphate (cAMP), Prolactin, Glucagon, niacinamide, ⁇ -lipoic acid, triiodothyronine, epidermal growth factor (EGF), high-density lipoprotein (HDL), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), or human growth hormone (HGH).
  • SCF stem cell factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • OSM oncostatin-M
  • TGF ⁇ 3, Jagged-1
  • the culture media in step (c) may comprise at least one of stem cell factor (SCF), fibroblast growth factor (FGF), interleukin-6 (IL-6), Fms-like tyrosine kinase 3 (FLT3), or leukemia inhibitory factor (LIF).
  • SCF stem cell factor
  • FGF fibroblast growth factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • LIF leukemia inhibitory factor
  • the liver organoid may be seeded with at least one of HSC obtained from cord blood, HSC obtained from bone marrow, HSC derived from induced pluripotent stem cells, HSC derived from embryonic stem cells, or HSC derived from direct reprogramming of adult somatic cells.
  • the differentiated hematopoietic cells may comprise hematopoietic progenitor cells (HPC), myeloid progenitor cells (MPC), lymphoid progenitor cells (LPC), lymphocytes, granulocytes, macrophages, erythrocytes, or platelets.
  • the differentiated hematopoietic cells may comprise an enriched population of differentiated erythrocytes.
  • the culture media in step (c) may comprise erythropoietin (EPO).
  • Bioscaffolds or “scaffolds” or “matrices” as used herein refer to a substrate on which cells can grow.
  • the bioscaffolds are derived from natural tissues or organs by decellularizing a natural tissue or a natural organ. These scaffolds are useful in both the medical and research settings.
  • the bioscaffolds disclosed herein may be used in hepatic tissue engineering, three-dimensional (3-D) cell culture systems and bioreactor systems.
  • bioscaffolds are comprised of highly conserved proteins and heavily cross-linked ECM components like collagens, elastin, fibronectin, laminin and proteoglycans, which retain the characteristic 3-D architecture of the natural tissue or organ.
  • a natural organ or a natural tissue is decellularized using a mix of detergents (e.g., Triton-X 100, SDS) and other chemicals (e.g., ammonium hydroxide, sodium deoxycholate) that allow retention of the native ECM and native vascular channels.
  • detergents e.g., Triton-X 100, SDS
  • other chemicals e.g., ammonium hydroxide, sodium deoxycholate
  • the terms “decellularized” and “acellular” refer to a natural organ or a natural tissue that has been manipulated to remove all or most of the cells from the organ or tissue that are not part of the native extracellular matrix (ECM) or native vascular channels.
  • ECM extracellular matrix
  • ECM Extracellular matrix
  • the ECM provides essential physical scaffolding for the cellular constituents of tissues and organs and also provides biochemical and biomechanical cues for tissue morphogenesis, differentiation and homeostasis.
  • the ECM obtained from a natural tissue or a natural organ that is generally chemically and physically unchanged following decellularization of the natural tissue or the natural organ is referred to herein as the "native ECM”.
  • micro-environment niche or “niche” are used to refer to a region in organ (e.g., fetal liver) that supports certain cellular functions of the organ. These niches are made up of one or more cell populations within the organ that produce chemical signals that influence cell function or behavior. For example, in the fetal liver, there are niches that support of HSC expansion (proliferation) or differentiation. The characteristics of a niche that supports HSC expansion are in part different from the characteristics of a niche that supports HSC differentiation. The cells in different niches communicate with one another through contact and/or release of paracrine factors to regulate the rate of HSC expansion and differentiation.
  • Natural organs and “natural tissues” are organs and tissues that are freshly retrieved from animals and humans without manipulation. Organs and tissues that may be used to carry out the present invention may be from any suitable animal source, including human, other mammalian (e.g., cat, dog, pig, cow, sheep, horse, monkey), avian ( e.g., chicken, turkey, duck, goose, etc.), reptile, amphibian, etc.
  • the natural organ is a liver and the natural tissue is a portion of a liver.
  • organoid or “organoid disc” or “disc” or “bioengineered organoid” are used to refer to an organ-like structure or a tissue-like structure that mimics some or all of the properties of a natural organ or natural tissue in vivo.
  • An organoid resembles a natural organ in structural appearance or qualities.
  • the organoid can resemble either an adult organ or a fetal organ in structural appearance or qualities.
  • the organoid retains the extracellular matrix and native vascular channels of the natural organ or natural tissue.
  • Organoids are derived in part from a natural organ or portion of natural tissue obtained from a donor subject.
  • the portion of tissue can range in size from at least 200,000 cells to nearly the full size of the natural organ.
  • An organoid can be derived from a full-size natural organ or portion of natural tissue.
  • the term organoid also refers to small excised portions of a larger organoid ( e.g., an excised disc).
  • the terms “subject”, “individual” and “patient” are used interchangeably to refer to an animal, preferably a human, but also includes other mammals (e.g., cat, dog, pig, cow, sheep, horse, monkey), birds ( e.g., chicken, turkey, duck, goose, etc.), reptiles, amphibians, etc.
  • mammals e.g., cat, dog, pig, cow, sheep, horse, monkey
  • birds e.g., chicken, turkey, duck, goose, etc.
  • reptiles e.g., amphibians, etc.
  • Native vascular channels refers to the circulatory system components present in natural organs and natural tissues, which include vessels and capillaries.
  • the vascular channels are distributed throughout natural organs and natural tissues and acts to provide nutrients and oxygen to the surrounding cells.
  • the vascular channels, not including the vascular cells, obtained from a tissue or organ that is generally unchanged following decellularization of the tissue or organ is referred to herein as native vascular channels.
  • liver organoids Disclosed herein are liver organoids, methods of making such organoids, and methods of using such organoids.
  • liver extracellular matrix (ECM)-derived scaffolds have been developed.
  • ECM extracellular matrix
  • a decellularized donor liver tissue is used as the bioscaffold for a liver organoid.
  • the bioscaffold has the native ECM and native vascular channels of the donor liver tissue.
  • Liver cells are then seeded onto the bioscaffold and cultured with culture media.
  • the liver cells may be hepatoblasts, stromal cells, cholangiocytes, endothelial cells, or any combination thereof.
  • a liver organoid comprising a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels; and liver cells having predominately fetal characteristics.
  • the liver cells may be immature or fetal-like in nature.
  • the liver cells may comprise at least one of liver progenitor cells, hepatoblasts, vascular cells, cholangiocytes, or stromal cells.
  • the vascular cells may comprise at least one of liver endothelial cells, liver sinusodial cells, vascular smooth muscle cells, or pericytes.
  • the hepatoblasts may comprise at least one of fetal liver hepatoblasts, hepatoblasts derived from adult-derived liver stem cells, hepatoblasts derived from induced pluripotent stem cells, or hepatoblasts derived from embryonic stem cells.
  • the liver cells may comprise an enriched fetal liver progenitor cell population comprising at least about 55-75 % hepatic cells and not more than about 15-25 % stromal cells and not more than about 5-15 % endothelial cells.
  • the liver cells may comprise an enriched fetal liver progenitor cell population comprising about 55-75 % hepatic cells, about 15-25 % stromal cells, about 5-15 % endothelial cells.
  • the liver cells may express at least one of ⁇ - fetoprotein or CYP3A7.
  • the hepatic cells may comprise fetal liver progenitor cells.
  • liver organoid comprising a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels; and liver cells having adult characteristics.
  • the liver cells may have mature or adult-like gene expression patterns or capabilities.
  • the liver organoid has liver cells that have fetal characteristics and adult characteristics, reflecting maturation of the cells of the organoid.
  • the liver cells comprise at least one of hepatoblasts, hepatocytes, vascular cells, cholangiocytes, or stromal cells.
  • the vascular cells may comprise at least one of liver endothelial cells, liver sinusodial cells, vascular smooth muscle cells, or pericytes.
  • the hepatocytes may be derived from at least one of fetal liver hepatoblasts, hepatoblasts derived from adult-derived liver stem cells, hepatoblasts derived from induced pluripotent stem cells, or hepatoblasts derived from embryonic stem cells.
  • the liver organoid may comprise hepatocytic cell clusters and biliary ducts.
  • the liver cells may express albumin.
  • the liver cells may express at least one cytochrome P450 isoform.
  • the liver cells may express at least one of CK7, CK19, transferrin, CYP3A4, HNF4 ⁇ , AST, ALT, TAT, CYP2E1 or A1AT.
  • the liver cells may synthesize urea.
  • the liver cells can metabolize certain drug compounds metabolized by the adult liver in vivo.
  • a method of generating a liver organoid comprising the steps of providing a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels, seeding the bioscaffold with liver cells; and culturing the liver cells with culture medium containing at least one growth factor to generate a liver organoid comprising differentiated liver cells over time.
  • the liver cells may comprise at least one of hepatoblasts, hepatocytes, vascular cells, cholangiocytes, or stromal cells.
  • the vascular cells may comprise at least one of liver endothelial cells, liver sinusodial cells, vascular smooth muscle cells, or pericytes.
  • the hepatoblasts may comprise at least one of fetal liver hepatoblasts, hepatoblasts derived from adult-derived liver stem cells, hepatoblasts derived from induced pluripotent stem cells, or hepatoblasts derived from embryonic stem cells.
  • the liver cells of step (b) may comprise an enriched fetal liver progenitor cell population comprising at least about 55-75 % hetpaticcells and not more than about 15-25 % stromal cells and not more than about 5-15 % endothelial cells.
  • the liver cells of step (b) may comprise an enriched fetal liver progenitor cell population comprising about 55-75 % hepatic cells, about 15-25 % stromal cells, about 5-15 % endothelial cells.
  • the hepatic cells may comprise fetal liver progenitor cells.
  • the liver organoid may comprise hepatocytic cell clusters and biliary ducts.
  • the differentiated liver cells of step (c) may express albumin.
  • the differentiated liver cells of step (c) may express at least one cytochrome P450 isoform.
  • the culture medium of step (c) may comprise oncostatin M.
  • the culture medium of step (c) may comprise at least one of dexamethasone, cyclic adenosine monophosphate (cAMP), Prolactin, Glucagon, niacinamide, ⁇ -lipoic acid, triiodothyronine, free fatty acids, epidermal growth factor (EGF), high-density lipoprotein (HDL), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), or human growth hormone (HGH).
  • cAMP cyclic adenosine monophosphate
  • Prolactin Prolactin
  • Glucagon Prolactin
  • niacinamide ⁇ -lipoic acid
  • triiodothyronine triiodothyronine
  • free fatty acids EGF
  • EGF epidermal growth factor
  • HDL high-density lipoprotein
  • HGF hepatocyte growth factor
  • FGF fibroblast growth factor
  • HGH human growth
  • liver cells used to generate the organoids are from natural sources (e.g., fetus tissue).
  • the liver cells from natural sources are enriched prior to culturing in the liver organoids.
  • Fetal liver cells can be identified by the expression of specific cell surface markers such as, e.g., EpCAM, ICAM1, ⁇ -fetoprotein, albumin (ALB), and CD18.
  • liver cells may be enriched by active selection of the fetal hepatic cell population using a cell surface marker characteristic of these cells.
  • stromal cells identified by, e.g., markers such as ⁇ SMA or CD105
  • endothelial cells identified by, e.g., markers such as CD31
  • the source of liver cells used to generate the organoids are is embryonic stem cells (ES cells).
  • ES cells are pluripotent stem cells that can be from the inner cell mass of a blastocyst, an early-stage embryo. ES cells can be induced to become liver cells (mature or immature) for use as described herein.
  • iPS cells are induced pluripotent stem cells (iPS cells or iPSC).
  • iPS cells are a type of pluripotent stem cell artificially derived from a non-pluripotent cell-typically an adult somatic cell-by inducing expression of specific genes (e.g., at least Oct-3/4 (Pou5f1), Sox2).
  • iPS cells are similar to natural pluripotent stem cells, such as embryonic stem (ES) cells, in many aspects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability.
  • ES embryonic stem
  • iPS cells can be generated from a variety of adult somatic cells, including, e.g., stomach cells, liver cells, skin cells and blood cells. iPS cells can be induced to become liver cells (mature or immature) for use as described herein.
  • liver cells used to generate the organoids are directly reprogrammed adult somatic cells.
  • Direct reprograming or transdifferentiation, is the direct conversion of one cell type to another without undergoing an intermediate pluripotent state or progenitor cell type.
  • Reprogramming of cells to a different cell type is usually done by either somatic cell nuclear transfer (SCNT) or through expression of transcription factors or microRNA precursors.
  • SCNT somatic cell nuclear transfer
  • Direct reprogramming can be performed using a variety of cells types, including, e.g., skin, muscle, blood, pancreatic, and neurons. Studies have shown that adult somatic cells (e.g., fibroblasts) can be directly reprogramed into multipotent blood progenitor cells [65].
  • the liver organoids are useful for studying liver development in vitro. Reconstitution of differentiation of fetal liver cells on bioscaffolds enables in vitro monitoring of different developmental stages with respect to morphology, molecular characteristics, and functionality.
  • the liver organoids present a small scale, simple system for early stage assessment of drugs without requiring use of animal studies.
  • the liver organoids are useful for in vitro assessment of pharmacokinetics and pharmacology drug metabolism and liver function.
  • the liver organoids are useful for in vitro toxicity studies. For example, in some examples, teratogenesis studies can be conducted using the liver organoids to assess the impact of compounds on morphogenesis during development/embryogenesis.
  • the types of studies described above are performed using liver organoids comprising liver cells having adult characteristics. In other examples, the studies may be performed using liver organoids comprising liver cells having predominately fetal characteristics. In other examples, the studies may be initiated on liver organoids comprising liver cells having predominately fetal characteristics and performed overtime to monitor the impact of the compounds assessed on maturation of the liver organoid to one comprising liver cells having adult characteristics. In certain examples, the types of studies described above are performed by introducing compounds to be assessed into the culture medium in which the liver organoids are being cultured.
  • liver organoids are produced as liver organoid discs.
  • liver ECM discs may be made from acellular animal livers (Panel A).
  • Human fetal liver cells enriched fetal liver progenitor cells
  • liver ECM discs may be seeded on the liver ECM discs and allowed to grow in culture ( e.g., 1 week).
  • H&E staining identifies formation of biliary ducts (Panel B) and clusters of hepatocytes/hepatoblasts (Panel C) after 3 weeks of culturing.
  • apical staining and membrane staining (Panel D), as well as IHC staining for CYP2A (Panel E) of biliary ducts with polarized epithelial can be performed after 3 weeks of culturing.
  • hepatocytes/hepatoblasts in the liver organoid can be assessed by staining for expression of albumin and AFP (Panel F).
  • Liver organoids are generated from acellular ECM discs seeded with human fetal liver cells (enriched fetal liver progenitor cells) (Panel A).
  • Biliary structure may be observed by H&E staining (Panel B) and also identified by HSC staining for bile ducts (ABST+) amongst hepatic cells (EpCAM+) (Panel D).
  • Hepatic structures may also be observed by H&E staining (Panel C) and identified by HSC staining for hepatic cell markers CYP2A (Panel E) and albumin and ⁇ -fetoprotein (Panel F).
  • fetal liver cells on the ECM discs into liver organoids occurs after 5-7 days of culturing.
  • the organoids have large expanses of liver cells (Panel A) that show hepatic morphology (Panel B) and also the formation of biliary duct structures (Panel C) as demonstrated by H&E staining.
  • the presence of hepatic and biliary cells present in the organoids can be confirmed by IHC staining for albumin and CK19, respectively.
  • the fetal liver cells seeded on the ECM discs are an enriched population of fetal liver progenitor cells (hFLPCs) isolated from fetal liver tissue.
  • hFLPCs fetal liver progenitor cells isolated from fetal liver tissue.
  • FACS analysis identifies the different subpopulations of cells present in the enriched hFLPCs, including hepatic cells (as shown by markers EpCAM, ICAM1, ⁇ -fetoprotein, albumin, and CK18), stromal cells (as shown by markers ⁇ -SMA and CD105), and endothelial cells (as shown by marker CD31).
  • the enriched hFLPCs contain about 55-75 % hepatic cells (primarily liver progenitor cells), about 15-25 % stromal cells, and about 5-15 % endothelial cells. In some examples, depending on the culture conditions used to generate the enriched hFLPCs, the enriched hFLPCs contain at least about 55-75 % hepatic cells (primarily liver progenitor cells), not more than about 15-25 % stromal cells, and not more than about 5-15 % endothelial cells.
  • the liver organoids can be used to assess liver function.
  • metabolic activity of liver organoid discs can be assessed as shown in Fig. 3 .
  • the liver organoid discs can be first incubated with phenobarbital and 3-MC in order to induce enzymatic activity and then incubated with Diazepam and 7-ethoxycoumarin. After 48 hrs, media samples can be collected and analyzed by mass spectrometry (Panels C and D), and compared with standards of individual metabolites (Panels A and B).
  • the media collected from the liver organoid discs can be assessed for Phase-1 metabolites (e.g., Temazepam, Nordiazepam and 7-hydroxycoumarin).
  • liver organoids can be used to assess liver function.
  • albumin and urea secretion by the hepatic cells in the organoids can be assessed as shown in Fig. 3E and Fig. 3F , respectively.
  • Fig. 11A A similar analysis is shown in Fig. 11A .
  • the liver organoids display characteristic expression of many liver cell markers. For example, as shown in Fig. 5A , RT-PCR analysis identifies increased expression of many liver proteins over time. In some examples, this expression greater than that observed for 2D monolayer cell cultures.
  • the liver organoids contain differentiated cell populations and more mature, adult-like morphology. For example, as shown by IHC in Fig. 5B , after 3 weeks of culturing, the liver organoids contain hepatic cell clusters (ALB+/CK19-) and biliary ductal structure (ALB-/CK19+) as can be shown by IHC analysis.
  • EpCAM expression decreases over time and expression moves from the cytoplasm at 1 week of culturing to the cell membrane after 3 weeks of culturing.
  • the organoid discs mature during culturing, showing progressive differentiation.
  • microarray analysis shows a gene expression pattern for putative liver genes more similar to adult than fetal human liver at 3 weeks (as shown in Fig. 6 ), as well as biliary and hepatic genes (as shown in Fig. 7 and Fig. 8 , respectively).
  • the liver organoids show developmental maturation over time while in culture. As shown in Fig. 9A , after 1 week of culture the liver organoids bear morphological and protein expression characteristics similar to fetal liver tissue, while after 3 weeks of culture, the bear morphological and protein expression characteristics similar to adult tissue as can be determined by IHC analysis. For example, in some examples, the liver organoids after 1 week of culture express ⁇ -fetoprotein (AFP)and low levels of albumin (ALB), while after and 3 weeks of culture do not express AFP but does express ALB fetal liver tissue, as do fetal and adult liver tissue, respectively. In some examples, as shown in Fig. 9B , the liver organoids have hepatic cell clusters characteristic of mature liver tissue.
  • AFP ⁇ -fetoprotein
  • ALB albumin
  • the hepatic clusters show expression of adult hepatocyte markers HNF4 ⁇ , alpha-1-antitrypsin (A1AT), and cytochrome P450 3A4 after three weeks of culture as can be shown by IHC analysis.
  • the liver organoids can be used to assess development of the liver.
  • the liver organoids express HNF4 ⁇ and AST after 3 weeks of culture.
  • the liver organoids showed high expression of different cytochrome P450 isoforms (mature liver enzymes) whether the organoids were induced with the drugs phenobarbital and 3-methylcholanthrene ( Fig. 10B ) or not ( Fig. 10C ).
  • the liver organoids are useful for assessing morphogenesis of liver structures during development.
  • the liver organoids show four different stages of bile duct formation (ductal layer, second ductal layer, immature duct, mature duct).
  • the liver organoids contain CK19+/EpCAM+/SOX9+ biliary duct cells identical to that observed during the fetal duct morphogenesis process including laminin in the basal membrane surrounding the CK19 + biliary duct cells (cholangiocytes) as shown in Fig. 12B (top row).
  • the liver organoids contain biliary structures exhibiting typical bile duct apical-basal polarity, indicated by the presence of primary cilia (antitubulin) and a bile salt transporter (ABST) in the apical membrane and beta-catenin on the baso-lateral membrane Fig. 12B (bottom row).
  • the liver organoids express of HNF6 (a critical transcription factor in bile duct morphogenesis), HNF1 ⁇ (a transcription factor important for lineage specification of hepatoblasts into cholangiocytes), anion exchange factor 2 (AE2); and GGT1, all markers of mature cholangiocytes.
  • a liver organoid can be created to mimic the function of the human fetal liver such that HSC can be cultured in vitro using the liver organoid to support the expansion and/or differentiation of HSC.
  • the disclosed invention allows for the in vitro expansion of long-term repopulating HSC in such a way that sufficient numbers of HSC can be obtained to engraft an adult, while simultaneously increasing committed progenitor populations that are able to shorten time to engraftment (i.e., speed hematopoietic recovery after transplant).
  • the described liver organoids, and methods of using them support asymmetric division and symmetric renewal of HSC (and HPC).
  • liver organoids are disclosed herein are able to (1) support HSC expansion on a large scale without sacrificing their self-renewal ability, (2) produce expanded HSC that are safe, transplantable, and free of feeder cells, serum proteins, and microbial agents, and (3) also support differentiation of HSC to allow large scale production of differentiated hematopoietic cells.
  • the availability of these liver organoids allows use of readily available sources of HSC that otherwise have limited utility (e.g., cord blood HSC) to use for transplantation to treat a range of illnesses.
  • these liver organoids permit the production of large pools of HSC having defined characteristics (e.g., blood type, HLA class) as well as specific HSC pools ( e.g., patient-specific, ethnicity matched).
  • the liver organoids also enable production of a wide range of differentiated hematopoietic cells.
  • liver organoid comprising:(a) a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels; and (b) liver cells comprising at least one micro- environment niche that supports hematopoietic stem cell (HSC) expansion or differentiation.
  • the liver organoids may comprise one or more types of liver cells.
  • the liver cells comprise at least two micro- environment niches, wherein in at least one micro-environment niche supports HSC expansion and at least one micro-environment niche supports HSC differentiation.
  • the liver cells of the liver organoid may comprise fetal liver cells.
  • the liver cells of the liver organoid may comprise at least one of liver progenitor cells, hepatoblasts, vascular cells, cholangiocytes, or stromal cells.
  • the vascular cells may comprise at least one of liver endothelial cells, liver sinusodial cells, vascular smooth muscle cells, or pericytes.
  • the stromal cells may comprise mesenchymal cells.
  • the hepatoblasts may comprise at least one of fetal liver hepatocytes, hepatoblasts derived from adult-derived liver stem cells, hepatoblasts derived from induced pluripotent stem cells, or hepatoblasts derived from embryonic stem cells.
  • the at least one micro-environment niche supports expansion and/or differentiation of HSC from a variety of sources. In some examples, the at least one micro-environment niche supports expansion or differentiation of at least one of HSC obtained from cord blood, HSC obtained from bone marrow, HSC derived from induced pluripotent stem cells, HSC derived from embryonic stem cells, or HSC derived from direct reprogramming of adult somatic cells. In certain examples, the at least one micro-environment niche supports expansion or differentiation of HSC obtained from cord blood.
  • the liver organoids provide a system to study the different micro-environment niches of the fetal liver that support asymmetric division and symmetric renewal of HSC.
  • a method of generating a liver organoid comprising the steps of: (a) providing a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels; (b) seeding the bioscaffold with liver cells; and (c) culturing the liver cells with the bioscaffold in the presence of culture media for sufficient time to produce at least one micro-environment niche in the bioscaffold that supports hematopoietic stem cell (HSC) expansion or differentiation.
  • the method generates liver organoids as described in the above examples.
  • the liver cells are cultured with the bioscaffold in the presence of culture media for sufficient time to produce at least two micro-environment niches in the bioscaffold, wherein at least one micro-environment niche supports HSC expansion and at least one micro-environment niche supports HSC differentiation.
  • the liver cells of step (a) may comprise fetal liver cells.
  • the liver cells of step (a) may comprise at least one of liver progenitor cells, hepatoblasts, vascular cells, cholangiocytes, or stromal cells.
  • the vascular cells may comprise at least one of liver endothelial cells, liver sinusodial cells, vascular smooth muscle cells, or pericytes.
  • the hepatoblasts may comprise at least one of fetal liver hepatoblasts, hepatoblasts derived from adult-derived liver stem cells, hepatoblasts derived from induced pluripotent stem cells, or hepatoblasts derived from embryonic stem cells.
  • the liver cells of step (a) may comprise an enriched fetal liver progenitor cell population comprising at least about 55-75 % hepatic cells and not more than about 15-25 % stromal cells and not more than about 5-15 % endothelial cells.
  • the liver cells of step (a) may comprise an enriched fetal liver progenitor cell population comprising about 55-75 % hepatic cells, about 15-25 % stromal cells, about 5-15 % endothelial cells.
  • the hepatic cells are primarily liver progenitor cells.
  • the culture media used the method may comprise components that facilitate development of the at least one micro-environment niche in the bioscaffold that supports HSC expansion or differentiation.
  • the culture media in step (c) may comprise at least one of stem cell factor (SCF), interleukin-6 (IL-6), or Fms-like tyrosine kinase 3 (FLT3), oncostatin-M (OSM), TGF ⁇ 3, Jagged-1, fetal bovine serum (FBS), dexamethasone, cyclic adenosine monophosphate (cAMP), Prolactin, Glucagon, niacinamide, ⁇ -lipoic acid, triiodothyronine, epidermal growth factor (EGF), high-density lipoprotein (HDL), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), or human growth hormone (HGH).
  • SCF stem cell factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • OSM oncostatin-M
  • TGF ⁇ 3, Jagged-1
  • the culture media in step (c) may contain factors that stimulate activation of endogenous Notch receptors, copper chelators, or enhancers of engraftment such as PEG2 or CXCR4.
  • factors that stimulate activation of endogenous Notch receptors, copper chelators, or enhancers of engraftment such as PEG2 or CXCR4.
  • no additional factors e.g., cytokines, growth factors
  • the at least one micro-environment niche supports expansion or differentiation of at least one of HSC obtained from cord blood, HSC obtained from bone marrow, HSC derived from induced pluripotent stem cells, HSC derived from embryonic stem cells, or HSC derived from direct reprogramming of adult somatic cells.
  • the liver cells seeded on the bioscaffold in step (b) are cultured in culture media comprising at least one of epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), glycogen synthase kinase-3beta inhibitor (GSK3 ⁇ i), or thiazovivin.
  • EGF epidermal growth factor
  • HGF hepatocyte growth factor
  • IGF-1 insulin-like growth factor 1
  • GSK3 ⁇ i glycogen synthase kinase-3beta inhibitor
  • thiazovivin thiazovivin.
  • the liver cells seeded on the bioscaffold in step (b) are cultured for about 5 days.
  • hematopoietic cells comprising the steps of: (a) obtaining a liver organoid comprising (i) a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels and (ii) liver cells comprising at least one micro-environment niche that supports hematopoietic stem cell (HSC) expansion or differentiation; (b) seeding the liver organoid with HSC; (c) culturing the HSC on the liver organoid with culture media; and (d) collecting expanded HSC and/or differentiated hematopoietic cells from the culture media.
  • the method uses liver organoids as described in the examples above.
  • the liver organoid comprises (i) a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels and (ii) liver cells comprising at least two micro-environment niches, wherein at least one micro-environment niche supports HSC expansion and at least one micro-environment niche supports HSC differentiation.
  • the liver cells of step (a) may comprise fetal liver cells.
  • the liver cells of step (a) may comprise at least one of liver progenitor cells, hepatoblasts, vascular cells, cholangiocytes, or stromal cells.
  • the vascular cells may comprise at least one of liver endothelial cells, liver sinusodial cells, vascular smooth muscle cells, or pericytes.
  • the hepatoblasts may comprise at least one of fetal liver hepatoblasts, hepatoblasts derived from adult-derived liver stem cells, hepatoblasts derived from induced pluripotent stem cells, or hepatoblasts derived from embryonic stem cells.
  • the liver cells of step (a) may comprise an enriched fetal liver progenitor cell population comprising at least about 55-75 % hepatic cells and not more than about 15-25 % stromal cells and not more than about 5-15 % endothelial cells.
  • the liver cells of step (a) may comprise an enriched fetal liver progenitor cell population comprising about 55-75 % hepatic cells, about 15-25 % stromal cells, about 5-15 % endothelial cells.
  • the hepatic cells are primarily liver progenitor cells.
  • the liver organoid can be seeded with HSC from a variety of sources.
  • the liver orgnaoid may be seeded with at least one of HSC obtained from cord blood, HSC obtained from bone marrow, HSC derived from induced pluripotent stem cells, HSC derived from embryonic stem cells, or HSC derived from direct reprogramming of adult somatic cells.
  • the liver organoid may be seeded with HSC obtained from cord blood.
  • the liver cells seeded on the bioscaffold in step (b) are cultured in culture media comprising at least one of epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), glycogen synthase kinase-3beta inhibitor (GSK3 ⁇ i), or thiazovivin.
  • the liver cells seeded on the bioscaffold in step (b) are cultured for about 5 days.
  • the culture media used the method may comprise components that facilitate development of the at least one micro-environment niche in the bioscaffold that supports HSC expansion or differentiation.
  • the culture media in step (c) may comprise at least one of stem cell factor (SCF), interleukin-6 (IL-6), or Fms-like tyrosine kinase 3 (FLT3), oncostatin-M (OSM), TGF ⁇ 3, Jagged-1, fetal bovine serum (FBS), dexamethasone, cyclic adenosine monophosphate (cAMP), Prolactin, Glucagon, niacinamide, ⁇ -lipoic acid, triiodothyronine, epidermal growth factor (EGF), high-density lipoprotein (HDL), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), or human growth hormone (HGH).
  • SCF stem cell factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • OSM oncostatin-M
  • TGF ⁇ 3, Jagged-1
  • the culture media in step (c) may contain factors that stimulate activation of endogenous Notch receptors, copper chelators, or enhancers of engraftment such as PEG2 or CXCR4. However, in some examples, no additional factors (e.g., cytokines, growth factors) are added to the culture media of step (c).
  • the culture media in step (c) may comprise at least one of stem cell factor (SCF), fibroblast growth factor (FGF), interleukin-6 (IL-6), Fms-like tyrosine kinase 3 (FLT3), or leukemia inhibitory factor (LIF).
  • SCF stem cell factor
  • FGF fibroblast growth factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • LIF leukemia inhibitory factor
  • the liver organoid may be seeded with at least one of HSC obtained from cord blood, HSC obtained from bone marrow, HSC derived from induced pluripotent stem cells, HSC derived from embryonic stem cells, or HSC derived from direct reprogramming of adult somatic cells.
  • the differentiated hematopoietic cells may comprise hematopoietic progenitor cells (HPC), myeloid progenitor cells (MPC), lymphoid progenitor cells (LPC), lymphocytes, granulocytes, macrophages, erythrocytes, or platelets.
  • the differentiated hematopoietic cells may comprise an enriched population of differentiated erythrocytes.
  • the culture media in step (c) may comprise erythropoietin (EPO).
  • a cell population comprising hematopoietic cells produced by a process comprising (a) obtaining a liver organoid comprising (i) a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels and (ii) liver cells comprising at least one micro-environment niche that supports hematopoietic stem cell (HSC) expansion or differentiation, (b) seeding the liver organoid with HSC, (c) culturing the HSC on the liver organoid with culture media; and (d) collecting hematopoietic cells from the culture media.
  • a liver organoid comprising (i) a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular channels and (ii) liver cells comprising at least one micro-environment niche that supports hematopoietic stem cell (HSC) expansion or differentiation
  • HSC hema
  • the hematopoietic cells may comprise expanded HSC and/or differentiated hematopoietic cells.
  • the organoid may comprise (i) a bioscaffold derived from a decellularized donor subject liver comprising a native extracellular matrix (ECM) and native vascular network and (ii) liver cells comprising at least two micro-environment niches, wherein at least one micro-environment niche supports HSC expansion and at least one micro-environment niche supports HSC differentiation.
  • the liver cells of step (a) may comprise fetal liver cells.
  • the liver cells of step (a) may comprise at least one of liver progenitor cells, hepatoblasts, vascular cells, cholangiocytes, or stromal cells.
  • the vascular cells may comprise at least one of liver endothelial cells, liver sinusodial cells, vascular smooth muscle cells, or pericytes.
  • the hepatoblasts may comprise at least one of fetal liver hepatoblasts, hepatoblasts derived from adult-derived liver stem cells, hepatoblasts derived from induced pluripotent stem cells, or hepatoblasts derived from embryonic stem cells.
  • the liver cells of step (a) may comprise an enriched fetal liver progenitor cell population comprising at least about 55-75 % hepatic cells and not more than about 15-25 % stromal cells and not more than about 5-15 % endothelial cells. In certain examples, the liver cells of step (a) may comprise an enriched fetal liver progenitor cell population comprising about 55-75 % hepatic cells, about 15-25 % stromal cells, about 5-15 % endothelial cells. In some examples, the hepatic cells are primarily liver progenitor cells.
  • the liver cells seeded on the bioscaffold in step (b) may be cultured in culture media comprising at least one of epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), glycogen synthase kinase-3beta inhibitor (GSK3 ⁇ i), or thiazovivin.
  • EGF epidermal growth factor
  • HGF hepatocyte growth factor
  • IGF-1 insulin-like growth factor 1
  • GSK3 ⁇ i glycogen synthase kinase-3beta inhibitor
  • thiazovivin thiazovivin.
  • the liver cells seeded on the bioscaffold in step (b) may be cultured for about 5 days.
  • the culture media in step (c) may exclude exogenous growth factors, and wherein expanded HSC and/or immature hematopoietic stem cells are collected in step (d).
  • the culture media in step (c) may comprise at least one of stem cell factor (SCF), fibroblast growth factor (FGF), interleukin-6 (IL-6), Fms-like tyrosine kinase 3 (FLT3), or leukemia inhibitory factor (LIF).
  • SCF stem cell factor
  • FGF fibroblast growth factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • LIF leukemia inhibitory factor
  • the culture media in step (c) may comprise at least one exogenous growth factor, and wherein differentiated hematopoietic stem cells are collected in step (d).
  • the culture media in step (c) may comprise at least one of stem cell factor (SCF), interleukin-6 (IL-6), Fms-like tyrosine kinase 3 (FLT3), oncostatin-M (OSM), TGF ⁇ 3, Jagged-1, fetal bovine serum (FBS), dexamethasone, cyclic adenosine monophosphate (cAMP), Prolactin, Glucagon, niacinamide, ⁇ -lipoic acid, triiodothyronine, epidermal growth factor (EGF), high-density lipoprotein (HDL), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), or human growth hormone (HGH).
  • SCF stem cell factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • OSM oncostatin-M
  • TGF ⁇ 3, Jagged-1
  • the culture media in step (c) may comprise at least one of stem cell factor (SCF), fibroblast growth factor (FGF), interleukin-6 (IL-6), Fms-like tyrosine kinase 3 (FLT3), or leukemia inhibitory factor (LIF).
  • SCF stem cell factor
  • FGF fibroblast growth factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • LIF leukemia inhibitory factor
  • the liver organoid may be seeded with at least one of HSC obtained from cord blood, HSC obtained from bone marrow, HSC derived from induced pluripotent stem cells, HSC derived from embryonic stem cells, or HSC derived from direct reprogramming of adult somatic cells.
  • the differentiated hematopoietic cells may comprise hematopoietic progenitor cells (HPC), myeloid progenitor cells (MPC), lymphoid progenitor cells (LPC), lymphocytes, granulocytes, macrophages, erythrocytes, or platelets.
  • the differentiated hematopoietic cells may comprise an enriched population of differentiated erythrocytes.
  • the culture media in step (c) may comprise erythropoietin (EPO).
  • the culture media used in the method may comprise components that facilitate development of the at least one micro-environment niche in the bioscaffold that supports HSC expansion or differentiation.
  • HSC i.e., expanded HSC
  • immature hematopoietic stem cells are collected from the culture media.
  • the culture media may exclude growth factors when expanded HSC and/or immature hematopoietic stem cells are collected from the culture media.
  • the culture media that excludes growth factors may comprise at least one of stem cell factor (SCF), fibroblast growth factor (FGF), interleukin-6 (IL-6), Fms-like tyrosine kinase 3 (FLT3), or leukemia inhibitory factor (LIF).
  • SCF stem cell factor
  • FGF fibroblast growth factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • LIF leukemia inhibitory factor
  • the culture media may contain factors that stimulate activation of endogenous Not
  • differentiated hematopoietic cells are collected from the culture media.
  • the differentiated hematopoietic cells comprise hematopoietic progenitor cells (HPC), myeloid progenitor cells (MPC), lymphoid progenitor cells (LPC), lymphocytes, granulocytes, macrophages, erythrocytes, or platelets.
  • the culture media may contain at least one growth factor when differentiated hematopoietic stem cells are collected in from the culture media.
  • the culture media comprising at least one growth factor may comprise at least one of stem cell factor (SCF), interleukin-6 (IL-6), or Fms-like tyrosine kinase 3 (FLT3), oncostatin-M (OSM), TGF ⁇ 3, Jagged-1, fetal bovine serum (FBS), dexamethasone, cyclic adenosine monophosphate (cAMP), Prolactin, Glucagon, niacinamide, ⁇ -lipoic acid, triiodothyronine, epidermal growth factor (EGF), high-density lipoprotein (HDL), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), or human growth hormone (HGH).
  • SCF stem cell factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • OSM Fms-like tyrosine kina
  • the culture media may contain at least one of stem cell factor (SCF), fibroblast growth factor (FGF), interleukin-6 (IL-6), Fms-like tyrosine kinase 3 (FLT3), or leukemia inhibitory factor (LIF).
  • SCF stem cell factor
  • FGF fibroblast growth factor
  • IL-6 interleukin-6
  • FLT3 Fms-like tyrosine kinase 3
  • LIF leukemia inhibitory factor
  • an enriched population of differentiated erythrocytes is collected.
  • differentiation of erythrocytes is promoted.
  • the culture media will at least include erythropoietin (EPO).
  • EPO erythropoietin
  • no additional factors e.g., cytokines, growth factors
  • the culture media does not include growth factors and/or cytokines.
  • the culture media is serum-free culture media.
  • both expanded HSC and differentiated hematopoietic cells
  • Some examples encompass use of factors in the culture media used in generating the liver organoids described herein and/or the culture media used in culturing HSC in the liver organoids described herein to produce hematopoietic cells (e.g., HSC and more differentiated hematopoietic cells).
  • Some extrinsic signaling molecules including colony-stimulating factors (CSFs) and interleukins (ILs) that activate intracellular signaling molecules such as kinases are known to influence HSC pluripotency, proliferation, and lineage commitment.
  • CSFs colony-stimulating factors
  • ILs interleukins
  • the culture media used to generate the liver organoids or the culture media used to produce hematopoietic cells may comprise one or more of these types of factors.
  • the different culture media described herein may comprise one or more cytokines, chemokines, receptor ligand molecules, or intracellular signaling molecules.
  • Proteins such as growth factors
  • other additives such as antibiotics, antiinflammatories, and modulators of the immune response
  • various treatments may be applied to enhance adherence of cells to the bioscaffold and/or to each other. Appropriate treatments are described, for example, in U.S. Patent No. 5,613,982 .
  • Such treatments include the application of various proteins, e.g., growth factors or extracellular matrix proteins to the bioscaffold substrate or to the growing construct. For example, collagen, elastin, fibronectin, laminin, or proteoglycans may be applied to the bioscaffold.
  • the bioscaffold can be impregnated with growth factors such as nerve growth factor (NGF), aFGF, bFGF, PDGF, TGF ⁇ , VEGF, GDF-5/6/7, BMP-1/2/3/4/5/6/7/13/12/14, IGF-1, etc., or these agents may be provided in the culture medium.
  • growth factors such as nerve growth factor (NGF), aFGF, bFGF, PDGF, TGF ⁇ , VEGF, GDF-5/6/7, BMP-1/2/3/4/5/6/7/13/12/14, IGF-1, etc., or these agents may be provided in the culture medium.
  • the liver organoid described herein, and methods relating thereto, are useful for in vitro culturing of HSC such that HSC can be expanded and/or differentiated using readily available sources of HSC.
  • the source of HSC for culturing is cord blood.
  • HSC may be obtained from other sources.
  • the source of HSC is bone marrow.
  • Bone marrow can be an appropriate source of HSC for culturing using the liver organoid described herein in instances where a subject has certain cancers of the blood or bone marrow (e.g., multiple myeloma or leukemia) and treatment of the subject with radiation or chemotherapy destroys the subject's immune system.
  • cancers of the blood or bone marrow e.g., multiple myeloma or leukemia
  • treatment of the subject with radiation or chemotherapy destroys the subject's immune system.
  • bone marrow Prior to treatment of the cancer, bone marrow can be harvested from the subject and subsequently cultured in vitro using the liver organoid and methods described herein.
  • the hematopoietic cells generated, particularly the HPC can then be transplanted back into the subject after the cancer treatment is completed.
  • HSC from natural sources is enriched prior to culturing in the liver organoids.
  • HSC and their differentiated progeny
  • CD Cluster of Differentiation
  • HSC may be enriched by active selection of the HSC cell population using a cell surface marker characteristic of HSC.
  • Human HSC are characterized by the following cell surface markers: aldehyde dehydrogenase high (ALDH(hi)), CD34+, CD133+, CD49F, CD59+, CD90/Thy1+, CD38low/(-), c-Kit(-)/low, and Lin(-).
  • ADH(hi) aldehyde dehydrogenase high
  • CD34+ CD133+
  • CD49F CD59+
  • CD90/Thy1+ CD38low/(-)
  • c-Kit(-)/low c-Kit(-)/low
  • Lin(-) Lin(-).
  • HSC are selected and/or enriched by selecting for cells expressing any one of these
  • the source of HSC is embryonic stem cells (ES cells).
  • ES cells are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage embryo. ES cells can be induced to become HPC for use as described herein.
  • the source of HSC is induced pluripotent stem cells (iPS cells or iPSC).
  • iPS cells are a type of pluripotent stem cell artificially derived from a non-pluripotent cell-typically an adult somatic cell-by inducing expression of specific genes (e.g., at least Oct-3/4 (Pou5f1), Sox2).
  • iPS cells are similar to natural pluripotent stem cells, such as embryonic stem (ES) cells, in many aspects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability.
  • ES embryonic stem
  • iPS cells can be generated from a variety of adult somatic cells, including, e.g., stomach cells, liver cells, skin cells and blood cells. iPS cells can be induced to become HPC for use as described herein. As the iPS cells can be obtained from a subject in need of a HSC transplantation, the HPC derived therefrom may result in improved transplantation outcome for the subject by reducing the risk of host-vs-graft disease.
  • the HSC are directly reprogrammed adult somatic cells.
  • Direct reprograming or transdifferentiation, is the direct conversion of one cell type to another without undergoing an intermediate pluripotent state or progenitor cell type.
  • Reprogramming of cells to a different cell type is usually done through expression of transcription factors or microRNA precursors.
  • Direct reprogramming can be performed using a variety of cells types, including, e.g., skin, muscle, blood, pancreatic, and neurons. Studies have shown that adult somatic cells (e.g., fibroblasts) can be directly reprogramed into multipotent blood progenitor cells [65].
  • liver organoids i.e., bioengineered human liver tissue constructs
  • bioengineered human fetal liver-like organoids tissue constructs
  • the liver organoids are built upon a natural 3-D liver extracellular matrix (i.e., bioscaffold), into which seeded liver cells (e.g., fetal liver-derived mesenchymal cells, hepatoblasts, and/or endothelial cells) become functionally integrated and assembled.
  • the bioscaffold contains necessary elements to mimic the fetal liver microenvironmental niches known to promote rapid expansion of HSC during development.
  • the liver organoids provide a model system in which to dissect the role of its individual cellular and matrix components in supporting CB maintenance, expansion, and differentiation.
  • a liver organoid may be produced that contains at least one microenvironment niche that supports HSC expansion or differentiation.
  • a decellularized donor liver tissue is used as the bioscaffold for a liver organoid.
  • the bioscaffold has the native ECM and native vascular channels of the donor liver tissue.
  • Liver cells are then seeded onto the bioscaffold and cultured with culture media.
  • the liver cells may be hepatoblasts, stromal cells, cholangiocytes, endothelial cells, or any combination thereof.
  • culturing the liver cells with the bioscaffold results in the production of a liver organoid comprising at least one micro-environment niche.
  • the liver organoid comprises at least two micro-environment niches-at least one niche that supports HSC expansion and at least one niche that supports HSC differentiation.
  • the liver organoid is seeded with HSC and the cells are cultured in the presence of culture media to support the growth of hematopoietic cells.
  • the cultured hematopoietic cells may be HSC, early progenitor cells such as HPC, MPC or LPC, or further differentiated cells such as the lymphocytes, granulocytes, and macrophages of the immune system, or erythrocytes and platelets. As show in Fig.
  • CD34 + CB-derived HSCs expand progressively and efficient on the organoids cultured with long term HSC.
  • HSCs may be expanded continuously for at least 7 weeks.
  • the liver organoid discs support expansion of mulipotent progenitor cells, as identified in colony-forming assays as CFU-GEMM.
  • the CFU-GEMM cell population is expanded 20-fold over 7 weeks of culturing.
  • the liver organoid discs supported maintenance of granulocyte, monocyte progenitors, as identified in colony-forming assays as CFU-GM for at least 6 weeks of culturing.
  • Liver Harvesting and Decellularization Four to five weeks old ferret livers (Marshall Bioresources, New York) are utilized for decellularization and disc preparation.
  • human, rodent e.g., rat, mouse
  • pig, monkey, ape, bovine and sheep livers can be used. Livers are harvested with intact vessels and the portal vein was cannulated with a 16-guage cannulae (Cathlon® Clear, Johnson & Johnson Medical Ltd., Arlington, TX, USA).
  • the livers are then connected to a pump (Masterflex L/S peristaltic pump with Masterflex L/S easy load pump head and L/S 14 gauge tubing, Cole-Palmer, Vernon Hills, IL, USA) and perfused with 2 liters of distilled water at the rate of 6 mL/min.
  • the livers are then perfused with 4 liters of detergent made up of 1% Triton-X 100 (Sigma-Aldrich Co., St. Louis, MO, USA) with 0.1% Ammonium hydroxide (Sigma-Aldrich Co., St. Louis, MO, USA).
  • the livers are perfused with 8 liters of distilled water to wash out the decellularization detergent.
  • FIG. 1A top left
  • Fig. 2A show images of decelluarlized ferret liver.
  • the cryopreserved liver lobes are mounted onto a cryotome (Leica CM1950 Crystat) to obtain liver ECM discs.
  • the cryotome temperature is set to about -8° C to -10° C in order to maintain the liver lobes at warmer temperatures, facilitating thick and intact sectioning of liver lobes.
  • the sections are cut to about 300 ⁇ m thickness.
  • an about 8 mm diameter biopsy punch equipped with a plunger is used in order to place the discs in the wells of, e.g., a 48-well plate.
  • the 48-well plate is kept inside the cryotome until the desired number of discs is generated.
  • the discs are then air-dried for up to 4-6 hours or until they are almost dry. Following the drying step, the discs are washed carefully with multiple washes of PBS and kept in PBS at 4° C until ready for sterilization.
  • the discs are sterilized by gamma irradiation at a dose of 1.5 Mrad using a Wheeler Cobalt-60 Gamma Irradiator.
  • hFLPCs Human fetal livers at developmental stages between approximately 18 and 21 weeks of gestation are obtained (e.g., Advanced Biological Resources, Alameda, CA). Detailed description for isolation of hFLPCs has been described previously [12, 78]. Briefly, nonhepatic tissue is removed by scalpel and livers are enzymatically digested at 37° C by collagenase type IV (Worthington Biochemical Corporation, New Jersey) and deoxyribonuclease to yield small tissue clumps.
  • a neutralizing wash using DMEM containing 10% FBS is performed, followed by two washed in Hepatocyte Washing Media (Gibco) (sedimentation at 1200-1500 g).
  • the resultant parenchymal cell clumps are resuspended in culture medium and then and passed through a 100 ⁇ m nylon mesh, followed by a 40 ⁇ m nylon mesh, to yield highly enriched suspensions of single cell and small cell aggregates of 3-8 hepatoblasts.
  • the cells are then resuspended in culture medium and then overlaid onto a Histopaque®-1077 density gradient (Sigma-Aldrich, 10771) and spun (400 g for 30 mintues) to separate the hematopoietic and nonparenchymal cells from the parenchymal cell fraction.
  • the resultant pellet is highly enriched in fetal hepatoblasts (single and small cell aggregates), liver stem cells, and red blood cells.
  • the interface between layers contains enriched amounts of non-parenchymal cells (stromal, endothelial).
  • the lower fraction cell pellet is resuspended in media and plated on Collagen-IV (5 mg/cm 2 ) and Laminin (1 mg/cm 2 ) coated 15-cm culture plates and incubated at 37° C. After 24 hours, the cells are washed to remove blood cells.
  • the enriched hFLPCs are cultured until enough cells are produced to seed onto acellular discs.
  • the enriched hFLPCs may also be cultured in suspension.
  • Different culture medias can be used to culture the enriched hFLPCs.
  • they can be cultured in liver progenitor cell (LPC) seeding medium as described in [12, 78] made of Advanced RPMI (Invitrogen, 12633012) containing 1% antibiotics/antimycotic (Invitrogen), 10 mg/L Ascorbic Acid, 0.04 mg/L dexamethasone, 2.45 mg/L cAMP, 10 ⁇ /L hProlactin, 1 mg/L hGlucagon, 10 mM niacinamide, 0.105 mg/L alpha lipoic acid, 67 ng/L triiodothyronine (Sigma-Aldrich), 40 ng/mL human Epidermal Growth Factor (hEGF) (R&D Systems, Inc., Minneapolis, MN, USA), 10 mg/L hHDL (Cell Sciences, Canton, MA, USA), 20 ng/mL human hepatocyte growth factor (hHGF)
  • the enriched hFLPCs can be cultured in minimal LPC media made of Kubota Medium (KM) containing RPMI 1640 containing 10 mg/L Ascorbic Acid, 0.04 mg/L Dexamethasone (or 10 -7 M hydrocortisone), 5 mM Niacinamide, 76 ⁇ l/L Free Fatty Acid Mix, 5 mg/L insulin, 10 mg/L transferrin, 5 ⁇ g/L sodium selenite and 0.5 g of bovine serum albumin (BSA), and supplemented with 10 ng/nL human Epidermal Growth Factor (hEGF) (R&D Systems, Inc., Minneapolis, MN, USA) and 10 ng/mL hepatocyte growth factor (HGF),.
  • KM Kubota Medium
  • RPMI 1640 containing 10 mg/L Ascorbic Acid, 0.04 mg/L Dexamethasone (or 10 -7 M hydrocortisone), 5 mM Niacinamide, 76 ⁇ l/L Free Fatty
  • KM is a serum-free and growth factor-free medium optimized for ex vivo expansion of hepatite progenitor cells [69].
  • they can be cultured in hepatic fetal liver progenitor (hFLP) medium made of KM supplemented with 10 ⁇ M thiazovivin, 20 ng/ml IGF-1 and 50 nM Glycogen Synthase Kinase-3beta inhibitor (GSK3 ⁇ i).
  • GSK3 ⁇ i Glycogen Synthase Kinase-3beta inhibitor
  • This protocol could be modified to isolate fetal liver mesenchymal stem cells (MSC) and hepatic sinusoidal endothelial cells (HSEC) from the pellet following density gradient separation using fluorescence-activated cell sorting with antibodies to Stro-1 and VE-cadherin and/or CD31. These markers have enabled consistent isolation of pure populations of MSC from a variety of tissues [32-36] and HSEC, respectively. These three populations can then be seeded onto the decellularized discs (e.g., 600,000 hepatoblasts, 200,000 MSC, and 200,000 HSEC per well/disc) for culturing as described below.
  • the decellularized discs e.g., 600,000 hepatoblasts, 200,000 MSC, and 200,000 HSEC per well/disc
  • hFLPCs are harvested from culture plates using collagenase IV and then counted.
  • hFLPCs (0.5 x 10 6 - 1.0 x 10 6 cells) are resuspended in 10 ⁇ l volume in medium for each disc.
  • the cell suspension (10 ⁇ l) is slowly pipetted on top of each disc and incubated for about an hour at 37° C for attachment before adding additional medium to a final volume of 500 ⁇ l (per well).
  • the same number of hFLPCs can be seeded on a collagen IV and laminin coated 48-well tissue culture plates.
  • the medium used for these steps can be either the LPC seeding medium or the minimal LPC medium.
  • liver differentiation medium made of Advanced RPMI (Invitrogen, 12633012) containing 1% antibiotics/antimycotic (Invitrogen), 10 mg/L Ascorbic Acid, 0.04 mg/L dexamethasone, 2.45 mg/L cAMP, 10 ⁇ /L hProlactin, 1 mg/L hGlucagon, 10 mM niacinamide, 0.105 mg/L alpha lipoic acid, 67 ng/L triiodothyronine (Sigma-Aldrich), 40 ng/mL human Epidermal Growth Factor (hEGF) (R&D Systems, Inc., Minneapolis, MN, USA), 10 mg/L hHDL (Cell Sciences, Canton, MA, USA), 20 ng/mL human hepatocyte growth factor (hHGF), 3.33 ng/mL human Growth Factor (hGH), 76 ⁇ l/L Free Fatty Acid Mix, and
  • Fig. 1 shows an overview of this process. Over a period of 3 weeks, a three dimensional organoid has formed through recellularization of the ECM disc.
  • Fluorescence activated cells sorting analysis of the enriched hFLPC population can be performed after 1 week of culturing.
  • Antibodies for different cell types can be used to assess the proportion of cells in the enriched population: putative hepatoblast markers (e.g ., EpCAM, ICAM1, ⁇ -fetoprotein ( ⁇ FP), ALB, CK18); stromal cells ( ⁇ SMA, CD105), endothelial cells (CD31).
  • putative hepatoblast markers e.g ., EpCAM, ICAM1, ⁇ -fetoprotein ( ⁇ FP), ALB, CK18
  • stromal cells ⁇ SMA, CD105
  • endothelial cells CD31
  • the enriched hFLPC population has about 55-75% hepatic cells (primarily liver progenitor cells), about 10-25% stromal cells, and about 5-15% endothelial cells when cultured using LPC seeding medium and LPC maintenance medium. Representative histograms are shown in Fig. 2G . Use of minimal LPC seeding medium and minimal LPC maintenance medium results in less differentiation of the initial cell populations (empirical analysis; data not shown).
  • hFLP medium to culture the enriched hFLPCs also results in less differentiation of the initial cell populations (less even than with the minimal LPC seeding and maintenance medias) and favors proliferation of the hepatic cells in the population over the stromal and endothelial cells (empirical analysis; data not shown).
  • the acellular discs were prepared using decelluarized ferret liver as described above in Section A ( Fig. 2A ).
  • the ECM discs were seeded with enriched hFLCPs and cultured as described above in Section A using LPC seeding and maintenance media.
  • the organoids can be prepared using minimal LPC medium or hFLP medium instead.
  • the seeded discs were cultured for 3 weeks and then harvesting for immunohistochemical and molecular analysis. Over the course of the incubation, the cells infiltrated the ECM and grew into 3-D liver organoids that formed biliary and hepatocytic structures like that of native liver ( Fig. 2B and 2C ), and expressed common hepatic markers (Fig.
  • bile duct-specific apical sodium dependent bile transporter ASBT
  • EpCAM EpCAM expressing hepatocytic cells
  • Fig. 2D Hepatocyte/hepatoblast clusters were also observed through the entire tissue expressing hepatic specific cytochrome P450 2A (CYP2A) ( Fig. 2E ), as well as albumin and ⁇ -fetoprotein (AFP) ( Fig. 2F ).
  • hepatocyte functional analysis culture medium was collected after the three week incubation. The media was stored at -80° C until it was used for analysis.
  • albumin ELISA assay (Bethyl Laboratories Inc.) was carried out on each samples in triplicates. The albumin concentrations were normalized per mg of DNA. The media was also analyzed in the same fashion for urea secretion using a QuantichromeTM Urea Assay Kit (BioAssay Systems, Inc.).
  • liver organoids were first incubated with phenobarbital and 3-MC in order to induce enzymatic activity and then incubated with diazepam and 7-ethoxycoumarin, two compounds known to be metabolized by liver enzymes. After 48 hours, media samples were collected and analyzed by mass spectrometry (Panels C and D), and compared with standards of individual metabolites (Panels A and B). After enzymatic activation, the media collected from the liver organoid discs was found to contain Phase-1 metabolites Temazepam, Nordiazepam and 7-hydroxycoumarin. These results support the conclusion that the engineered liver tissue is functional, over a long period of time (3 weeks), and possess metabolic capabilities of a native human liver.
  • the seeded fetal liver cells became fully functional upon integration into the scaffold, secreting albumin and urea ( Fig. 3E and 3F ), and acquiring the ability to correctly metabolize common drugs such as diazepam and 7-ethoxycoumarin ( Fig. 3A-D ), which are functionalities that fetal livers have, though to a lesser extent that adult liver.
  • common drugs such as diazepam and 7-ethoxycoumarin
  • Fig. 3A-D are functionalities that fetal livers have, though to a lesser extent that adult liver.
  • the degree of differentiation or function of these fetal liver cells will be manipulated using culture conditions. To generate a more immature liver organoid, culture media containing very few growth factors will be used ( e.g ., selected from those described herein, amongst others).
  • the liver organoids were prepared as described above in Example 1, Section A using LPC seeding and maintenance media.
  • the organoids can be prepared using minimal LPC medium or hFLP medium instead.
  • the discs were cultured for 3 weeks, with harvesting of cells at 1 week and 3 weeks for immunohistochemical and molecular analysis.
  • Assays assessing albumin and urea synthesis and IHC analyses were performed as described above.
  • Gene expression analyses were performed by RT-PCR after harvesting the organoids. Drug metabolism activity in the organoids was assessed after 1 week and 3 weeks of culturing.
  • liver organoids were first incubated with phenobarbital and 3-MC in order to induce enzymatic activity and then incubated with diazepam and 7-ethoxycoumarin. After 3, 6, 12, and 24 hours, media samples were collected and analyzed by mass spectrometry. After enzymatic activation, the media collected from the liver organoid discs was assessed for the presence of Phase-1 metabolites Temazepam, Nordiazepam and 7-hydroxycoumarin.
  • RT-PCR analysis was used to assess expression of genes characteristic of biliary cells (CK7, CK19), hepatic cells (ALT, AST), hepatoblasts (AFP, ALB, CYP3A7), hepatocytes (transferrin), and proliferating cells (Ki-67). This analysis showed more extensive differentiation, proliferation and higher survival of hFLPCs in the 3D liver organoids than in 2D tissue culture dishes coated with collagen IV and laminin ( Fig. 5A ).
  • GSEA Gene Set Enrichment Analysis
  • liver genes of interest e.g., liver progenitor, hepatic, biliary, carbohydrate metabolism, drug metabolism, fatty acid metabolism), reactome synthesis of bile acids and bile salts, mature liver cells (e.g., hepatocyte and biliary).
  • the genes in these panels are listed at the end of this section. Expression in liver organoid discs after 1 week or 3 weeks culturing was compared to expression in adult and fetal tissue as well as whole recellularized organ prepared as described in [12, 78].
  • Hepatocyte maturation is a dynamic process highlighted by changes in levels of various cytokines and transcription factors associated with differentiation and maturation of hepatoblast into hepatocytes.
  • Transcriptional switch from ⁇ -fetoprotein (AFP) to albumin resulting into loss of AFP expression and increased levels of albumin is one of the hallmark of hepatocyte maturation.
  • hFLPCs displayed this progressive maturation into hepatocytes within the organoids as clusters of cells expressed both AFP and albumin after 1 week of differentiation, similar to fetal liver ( Fig. 9A , left panel), and subsequently lost AFP expression as the cells matured within the organoids after 3 weeks of differentiation, resembling adult liver ( Fig. 9A , right panel).
  • hepatocyte markers such as HNF4 ⁇ , alpha-1-antitrypsin (A1AT) and cytochrome P450 3A4 ( Fig. 9B ).
  • RT-PCR analysis further confirmed expression of hepatocyte differentiation markers including HNF4 ⁇ and AST ( Fig. 10A ).
  • Differentiated hepatocytes also showed high expression of different cytochrome P450 isoforms whether they were induced with the drugs phenobarbital and 3-methylcholanthrene ( Fig. 10B ) or not ( Fig. 10C ).
  • the liver organoids also showed significantly higher albumin and urea secretion compared with hFLPCs differentiated in culture plates ( Fig. 11A ).
  • the liver organoids metabolized diazepam into temazepam and nordiazepam, and 7-ethoxy coumarin into 7-hydroxy coumarin, both phase I metabolites ( Fig . 11B ).
  • liver progenitor cells also differentiate into cholangiocytes that give rise to bile ducts through unique morphogenesis. This process lacks in many of the in vitro liver organoid systems but the liver organoid system described above allows for concomitant bile duct morphogenesis.
  • Four different stages of bile duct formations were observed in these organoids, comparable to the duct developmental stages observed in human fetal liver ( Fig. 12A ).
  • the ECM composition around these biliary duct cells was identical to that observed during the fetal duct morphogenesis process including laminin in the basal membrane surrounding the CK19 + biliary duct cells (cholangiocytes).
  • RT-PCR analysis also showed up-regulation of HNF6 (a critical transcription factor in bile duct morphogenesis), HNF1 ⁇ (a transcription factor important for lineage specification of hepatoblasts into cholangiocytes), anion exchange factor 2 (AE2); and GGT1, all markers of mature cholangiocytes ( Fig. 12C ).
  • HNF6 a critical transcription factor in bile duct morphogenesis
  • HNF1 ⁇ a transcription factor important for lineage specification of hepatoblasts into cholangiocytes
  • anion exchange factor 2 AE2
  • GGT1 all markers of mature cholangiocytes
  • liver organoid Preparation The liver organoid system can be modified to support the maintenance and expansion of HSPC. Fetal livers are harvested, decelluarized, acellular ECM discs are prepared, and enriched hFLPCs are isolated as described above in Example 1 with the following modifications. Sterilized discs were incubated with KM medium for 30-45 minutes prior to cell seeding and then air dried in biosafety cabinet. hFLPCs were re-suspended in hFLP medium, and each disc was seeded with 0.5 x 10 6 hFLPCs in hFLP medium. Alternatively, the hFLPCs can be resuspended in Kubota's Media (KM) and seeded in minimal LPC medium. These seeded discs were then allowed to mature for ⁇ 5 days at 37° C to allow the formation of functional liver organoids. The culture medium is changed every 24 hours.
  • KM Kubota's Media
  • CB Cord blood
  • Cord blood (CB) units are obtained from publicly available sources (e.g., NHLBI BioLINCC Biologic Specimen and Data Repository Information Coordinating Center). Each CB unit is expected to contain about 200 x 10 6 total cells but some CB units will have significantly fewer cells and other CB units will have significantly more cells. CB units are thawed, and the mononuclear fraction obtained by centrifugation over a Ficoll density gradient. If necessary, the mononuclear cell fraction is incubated with an ammonium chloride solution (STEMCELL Technologies) to lyse any residual red blood cells.
  • an ammonium chloride solution STMCELL Technologies
  • Magnetic separation using MiniMACSTM columns is performed to obtain an enriched population of CD34+ HSP cells.
  • Use of MiniMacs columns to enrich for CD34+ cells enables enrichment of small numbers of highly primitive CD34(-) HSC found in CB units that are often removed during high stringency sorting, resulting in a reduced output from feeder-based expansion cultures [3, 4, 6, 8].
  • HSPC enrichment may alternatively be performed by lineage depletion rather than positive selection for CD34 expression.
  • Flow cytometry FACS analysis
  • FACS analysis is performed to determine the purity of the isolated HSPC population as described in [3, 4, 6, 8, 37].
  • LTC-IC Long term culture-initiating cells
  • CAFC cobblestone area-forming cells
  • HSPC Culturing on Organoids After incubating the organoids for 5-7 days, 125,000 CD34 + HSP cells were seeded onto each disc. The following different culture media conditions were tested in triplicate: (1) KM, (2) hFLP growth medium; and (3) long term HSC medium made of QBSF-60 serum-free medium (Quality Biological, Gaithersburg, MD, USA) containing 100 ng/ml stem cell factor (SCF), 5 ng/ml basic fibroblast growth factor (bFGF), 10 U/mL leukemia inhibitory factor (LIF), and 100 ng/mL Flt-3 (Peprotech, Rocky Hill, NJ, USA) [3, 4]. Each organoid was cultured at 37° C in a final volume of 1 mL medium.
  • SCF stem cell factor
  • bFGF basic fibroblast growth factor
  • LIF leukemia inhibitory factor
  • Flt-3 Flt-3
  • the liver organoids were fixed with 10% buffered formalin (Fisher Scientific, Inc., USA), tissue processed and paraffin embedded. Blocks were sectioned at 5 ⁇ m and antigen retrieval was performed using Target Retrieval Solution (Dako Co, USA). Immunohistochemical (IHC) analysis was then performed for using primary antibodies for EpCAM (Santa Cruz Biotechnology, USA), CK19 (Santa Cruz Biotechnology, USA), CD45 (BD, USA), ⁇ SMA (Abcam, UK) and Hemoglobin F (Bethyl Laboratories, USA). Appropriate secondary antibodies were used for imaging. Sections were analyzed and pictures were taken with a fluorescence microscope (Carl Zeiss Gmgb, Germany).
  • IHC analysis showed that there is extended in vitro survival of abundant clusters of EpCAM+ hepatic cells in the bioengineered liver tissue ( Fig. 14A ) compared to what is observed under the culture conditions described in Example 1, Section B (growth up to 4 weeks). IHC analysis also showed several CD45+ hematopoietic cells surrounding EpCAM+ hepatic cell clusters and aSMA+ stromal cells, showing two potential stromal populations of the bioengineered liver organoids ( Fig. 14B-D ).
  • HSCs isolated from cord blood were also assessed directly in the colony forming assay without first culturing on the organoids to determine the baseline colony forming potential of the cell population.
  • CD34 + CB-derived cells expanded progressively and more efficiently in the wells containing long term HSC media ( Fig. 15A ). Neither KM nor hFLP medium supported expansion of the CD34 + cells. Significantly, the CD34 + cells were expanded continuously for all 7 weeks of the analysis. This is in contrast to previous studies where HSP cells were cultured using the same long term HSC medium on stromal cells as a feeder layer and were found to expand efficiently for only the first 2 weeks of culturing [3-4].
  • CFU-GEMM assess the presence of multipotent progenitor cells that give rise to granulocytes, erythrocytes, monocytes, and megakaryocytes. These cells give rise to CFU-GM and, eventually, to BFU-E cell populations.
  • CFU-GM assess the presence of granulocyte and monocyte progenitors, which give rise to monoblasts and myeloblasts.
  • BFU-E assess the presence of the earliest known erythroid precursor cell, which give rise to proerythroblasts.
  • Colony-forming assays demonstrated that, during the first week of culture, expansion occurred within the most primitive hematopoietic cells as observed by the 20-fold expansion of CFU-GEMM and that, by week 4, these cultures still contained significant numbers of these primitive colonies ( Fig. 15B ). The final accumulated number of CFU-GEMM throughout the 7 weeks of the experiment totaled a 25-fold expansion. Also, in comparison with our previously published work in which CFU-GM were lost from culture after 2 weeks, CFU-GM were further maintained in culture until week 6 by culturing on the fetal liver organoid [3-4].
  • Organoids can be prepared as described above in Example 2, Section A. As references/controls, as described in [4], the following will be included: 1) cultures in which these three cell populations are plated at this same ratio directly into tissue culture wells to grow in 2-D monolayers; and 2) monolayer cultures of bone marrow-derived MSC (BM-MSC).
  • HSPCs will be prepared from CB as described above in Example 2, Section A. Once the organoids have fully repopulated and the monolayers have been established, culturing of CB-HSPCs will begin.
  • ⁇ 250,000 CB-HSPC will be plated in each well containing a hepatic organoid, a 3 cell type monolayer (control), or a BM-MSC monolayer (control), and various media conditions will be tested on the 5-7 day organoids as described above.
  • One condition will be the long term HSC medium.
  • Another condition will include supplementing the medium with a cytokine cocktail 20 ng/mL SCF, 10 ng/mL IL6, and 30 ng/mL FLT3 previously employed in hepatoblast-based HSPC expansion cultures as described in [18].
  • Other media conditions will also be evaluated, removing or adding certain components.
  • organoids will be grown in medium with no added cytokines to rigorously test the ability of the fetal liver organoids to support CB-derived hematopoiesis. While other culture media conditions will be assessed (e.g., containing other components to stimulate HSC expansion), no growth factors will generally be included in the culture media so as to avoid stimulating differentiation of the HSC into mature hematopoietic stem cells. Every 12 hours, 0.5 mL of fresh media will be added to wells. See [39] (indicating that frequent media addition is required to continually dilute factors that cause inhibitory feedback and thus block subsequent HSPC expansion).
  • hematopoietic stem/progenitor activity present within the various expansion cultures cells collected from the organoid cultures each 72 hours will be analyzed using traditional, methylcellulose colony-forming assays (CFU-GEMM, BFU-E, and CFU-GM) to assess the progenitor content of the expanded hematopoietic cell population [25].
  • CFU-GEMM, BFU-E, and CFU-GM methylcellulose colony-forming assays
  • HuSPCA Human Stem/Progenitor Cell Assay
  • HPP-CFC HighP-CFC
  • CFC-GEMM CFC-GM
  • BFU-E CFC-Mk
  • CFC-T CFC-T
  • CFC-B CFC-B.
  • HALO® relies upon advanced, ATP-based, luciferin/luciferase bioluminescence readout.
  • the assay is highly reproducible, eliminates the inherent subjectivity of visually-scored methylcellulose assays, and is extremely sensitive, requiring roughly 2-logs fewer input cells and allowing the detection of as little as 20 cells.
  • LTC-IC culture-initiating cell
  • liver scaffolds The availability of decellularized liver scaffolds and the ability to selectively repopulate these scaffolds with specific cell populations enables systematic study of the role each niche cell type plays in CB-HSPC fate determination. These studies will allow optimization of conditions that favor either HSC expansion or HSC differentiation. Liver organoids will be prepared as described in Example 2, Section A and B, using long term HSC medium and including any modifications deemed appropriate based on Example 2, Section C and cultured with CD34+ HSPC prepared as described in Example 2, Section A. A control group of empty scaffolds (no cells) will be included to account for the effect of the ECM alone.
  • the ECM is now appreciated to be a key regulator of cell and tissue behavior, by virtue of its ability both to arrange cells in a highly ordered 3-D array, and to serve as a reservoir of growth factors and cytokines [47-49]. As such, it is expected that the fetal liver ECM will exert some effects upon CB-HSPC, even in the absence of niche cells.
  • CB-HSPC will be plated in each well containing (i) an acellular hepatic disc, or (ii) an acellular hepatic disc repopulated with enriched hFLPCs (or isolated populations of hepatic, stromal and endothelial cells) and the media will be changed long term HSC medium as described above.
  • hFLPCs or isolated populations of hepatic, stromal and endothelial cells
  • Additional culture media conditions will include supplementing the basic culture media and the cytokine culture media with one or more growth factors, hormones, or other factors to support HSC differentiation.
  • Some growth factors, hormones, or other factors that will be tested include oncostatin-M (OSM), TGF ⁇ 3, Jagged-1, fetal bovine serum (FBS), dexamethasone, cyclic adenosine monophosphate (cAMP), Prolactin, Glucagon, niacinamide, ⁇ -lipoic acid, triiodothyronine, epidermal growth factor (EGF), high-density lipoprotein (HDL), hepatocyte growth factor (HGF), or human growth hormone (HGH).
  • OSM oncostatin-M
  • TGF ⁇ 3, Jagged-1 fetal bovine serum
  • cAMP cyclic adenosine monophosphate
  • Prolactin Prolactin
  • Glucagon Prolactin
  • Glucagon niaci
  • the decellularized hepatic scaffold discs, repopulated with enriched hFLPCS (or isolated hepatoblasts, MSC, and HSEC, singly or in combination) as described above provide an ideal system to study the micro-environment niches of the fetal liver that support asymmetric division and symmetric renewal of HSC.
  • Asymmetric division is regulated by cell polarity, with specific components of the cell membrane, cytoplasmic constituents, and even nuclear contents being unevenly distributed throughout the stem cell such that, upon division, two daughter cells of differing composition are created [50-52].
  • One key membrane protein within HSC that has been shown to segregate during cell polarization is CD133 [53].
  • CD133 + cells from cord blood and bone marrow are highly enriched in long term culture-initiating cells (LTC-IC) [55, 56] and long-term repopulating cells [57, 58], and expression of CD133 is lost upon differentiation.
  • LTC-IC long term culture-initiating cells
  • 57, 58 long-term repopulating cells
  • CD133 is lost upon differentiation.
  • the transfer of CD133 to daughter progeny of CB-HSPC expanded in the various hepatic discs will be used to determine the characteristics of cellular niches that support asymmetric division and the characteristics of cellular niches that promote self-renewing symmetric division.
  • Fetal liver organoids will be prepared as described above in Example 2, Sections A-C and culture conditions determined by the experiments described in Example 3 to induce: 1) the greatest degree of expansion of long term culture-initiating cells (LTC-IC); or 2) maintenance of LTC-IC numbers with concomitant production of committed progeny. These outcomes should be indicative of symmetric renewal and asymmetric division, respectively.
  • cord blood mononuclear cells will be labeled with the fluorescent membrane dye PKH2 (as described in [59]), and then PKH2-labeled CD133 + HSPC will be isolated on a FACS Aria II, depositing single cells into the individual disc-containing wells of the 96-well plates.
  • the niche cells may communicate with one another through contact and/or release of paracrine factors, and may thus exert greater, synergistic effects when working together.
  • the outcome of the expansion cultures in which the liver scaffold has been repopulated with a single niche cell type may differ from that obtained when all three cell types are seeded into the scaffold together. Differences such as degree of expansion, extent of differentiation, rate of differentiation, and skewing of lineage output would all provide valuable information about the role each cell type within the niche likely plays in the process of fetal liver hematopoiesis and CB-HSPC expansion.

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Claims (14)

  1. Procédé de production d'un organoïde de foie, comprenant les étapes consistant:
    (a) à fournir un bioéchaffaudage issu d'un foie de sujet donneur décellularisé comprenant une matrice extracellulaire (ECM) native et des canaux vasculaires natifs;
    (b) à ensemencer le bioéchaffaudage avec des cellules hépatiques foetales, lesquelles cellules hépatiques foetales comprennent au moins l'un parmi des hépatoblastes ou des cellules stromales exprimant CD105 ou alpha-SMA; et
    (c) à cultiver les cellules hépatiques avec un milieu de culture contenant au moins un facteur de croissance pendant un temps suffisant pour produire un organoïde de foie comprenant au moins l'un parmi une niche micro-environnementale qui supporte l'expansion des cellules souches hématopoïétiques (HSC) ou une niche micro-environnementale qui supporte la différenciation des HSC.
  2. Procédé selon la revendication 1, dans lequel les cellules hépatiques sont cultivées avec le bioéchaffaudage en présence d'un milieu de culture pendant un temps suffisant pour produire au moins une niche micro-environnementale qui supporte l'expansion des HSC et au moins une niche micro-environnementale qui supporte la différenciation des HSC.
  3. Organoïde de foie, consistant en:
    (a) un bioéchaffaudage issu d'un foie de sujet donneur décellularisé comprenant une matrice extracellulaire (ECM) native et des canaux vasculaires natifs; et
    (b) des cellules hépatiques foetales, lesquelles cellules hépatiques foetales comprennent au moins l'un parmi des hépatoblastes ou des cellules stromales exprimant CD105 ou alpha-SMA,
    lequel organoïde de foie comprend au moins l'une parmi une niche micro-environnementale qui supporte l'expansion des HSC ou une niche micro-environnementale qui supporte la différenciation des HSC, et lequel organoïde de foie est obtenu par le procédé selon la revendication 1 ou 2.
  4. Organoïde de foie selon la revendication 3, lequel organoïde de foie comprend au moins une niche micro-environnementale qui supporte l'expansion des HSC et au moins une niche micro-environnementale qui supporte la différenciation des HSC.
  5. Procédé selon la revendication 1 ou 2, ou organoïde de foie selon les revendications 3 ou 4, dans lequel ladite au moins une niche micro-environnementale supporte l'expansion ou la différenciation d'au moins l'une parmi des HSC obtenues à partir de sang de cordon, des HSC obtenues à partir de moelle osseuse, des HSC issues de cellules souches pluripotententes induites, des HSC issues de cellules souches embryonnaires ou des HSC issues du reprogrammage direct de cellules somatiques adultes.
  6. Procédé selon l'une quelconque des revendications 1, 2 ou 5, dans lequel le milieu de culture comprend au moins l'un parmi le facteur de croissance épidermique (EGF), le facteur de croissance des hépatocytes (HGF), le facteur de croissance analogue à l'insuline 1 (IGF-1), le bêta inhibiteur de la glycogène synthase kinase-3 (GSK3βi) ou la thiazovivine, et dans lequel de préférence les cellules hépatiques ensemencées sur le bioéchaffaudage dans l'étape (b) sont cultivées pendant environ 5 jours.
  7. Procédé de production de cellules hématopoïétiques (HSC), comprenant les étapes consistant:
    (a) à obtenir un organoïde de foie selon la revendication 3 ou 4 ou à mettre en oeuvre le procédé selon l'une quelconque des revendications 1, 2, 5 ou 6;
    (b) à ensemencer l'organoïde de foie avec des HSC;
    (c) à cultiver les HSC sur l'organoïde de foie avec un milieu de culture; et
    (d) à recueillir les HSC expansées et/ou les cellules hématopoïétiques différenciées à partir du milieu de culture.
  8. Procédé selon la revendication 7, dans lequel le milieu de culture dans l'étape (c) exclut les facteurs de croissance exogènes, et dans lequel les HSC expansées et/ou les cellules souches hématopoïétiques immatures sont recueillies dans l'étape (d).
  9. Procédé selon la revendication 8, dans lequel le milieu de culture dans l'étape (c) comprend au moins l'un parmi le facteur des cellules souches (SCF), le facteur de croissance des fibroblastes (FGF), l'interleukine-6 (IL-6), la tyrosine kinase 3 analogue à Fms (FLT3) ou le facteur inhibiteur de leucémie (LIF).
  10. Procédé selon la revendication 7, dans lequel le milieu de culture dans l'étape (c) comprend au moins un facteur de croissance exogène, et dans lequel les cellules souches hématopoïétiques différenciées sont recueillies dans l'étape (d).
  11. Procédé selon la revendication 10, dans lequel le milieu de culture dans l'étape (c) comprend au moins l'un parmi le facteur des cellules souches (SCF), l'interleukine-6 (IL-6), la tyrosine kinase 3 analogue à Fms (FLT3), l'oncostatine M (OSM), le TGFβ3; Jagged-1, le sérum de veau foetal (FBS), la dexaméthasone, l'adénosine monophosphate cyclique (cAMP), la Prolactine, le Glucagon, le niacinamide, l'acide α-lipoïque, la triiodothyronine, le facteur de croissance épidermique (EGF), une lipoprotéine de haute densité (HDL), le facteur inhibiteur de leucémie (LIF), le facteur de croissance des hépatocytes (HGF), le facteur de croissance des fibroblastes (FGF) ou l'hormone de croissance humaine (HGH).
  12. Procédé selon l'une quelconque des revendications 7 à 11, dans lequel l'organoïde de foie est ensemencé avec au moins l'une parmi des HSC obtenues à partir de sang de cordon, des HSC obtenues à partir de moelle osseuse, des HSC issues de cellules souches pluripotententes induites, des HSC issues de cellules souches embryonnaires ou des HSC issues du reprogrammage direct de cellules somatiques adultes.
  13. Procédé selon l'une quelconque des revendications 7, 10 ou 11, dans lequel les cellules hématopoïétiques différenciées comprennent les cellules progénitrices hématopoïétiques (HPC), les cellules progénitrices myéloïdes (MPC), les cellules progénitrices lymphoïdes (LPC), les lymphocytes, les granulocytes, les macrophages, les érythrocytes ou les plaquettes.
  14. Procédé selon la revendication 7, dans lequel le milieu de culture dans l'étape (c) comprend de l'érythropoïétine (EPO), et dans lequel les cellules hématopoïétiques différenciées comprennent une population enrichie d'érythrocytes différenciés.
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